6.a. 1.1. Introductory Overview Sequence databases and technology development that emanated from the Human Genome Project now offer avenues of molecular genetic and functional genomic analyses in biomedical research which were previously impossible as recently as the previous funding cycle for this Center Grant. The rapid pace of technological and instrumentation improvements that facilitate cutting edge molecular genetic and genomic approaches requires that we acquire new technologies, and have a highly skilled support staff that is adept at developing and supporting new instrumentation and methodologies. The Molecular Genetics Core has evolved to advance MRDDRC related research projects in three key areas: 1) high-throughput sequence analysis, 2) genomewide genetic marker genotype analysis for mapping and positional cloning, and 3) array-based transcriptional profiling. In providing these services to MRDDRC investigators, the Molecular Genetics Core has developed a cost-effective and efficient mechanism for these technically sophisticated approaches to be centrally managed to maintain quality and speed of data turn-around to MRDDRC investigators. The three functional components of the Molecular Genetics Core - Sequencing, Genotyping, and Expression Arrays - are all heavily utilized by MRDDRC investigators. Automated sequencing was established in the Core in the late 1980's following the cloning of the dystrophin locus, which ultimately led to the complete sequence analysis of three autosomal recessive forms of dystrophy. In its current form, the Core is being utilized in a high throughput sequencing strategy to identify mutations in all the known genes mutated in muscle diseases. Additionally, numerous MRDDRC investigators submit DMA for sequencing in the Core rather than outsourcing projects to commercial vendors because the in-house service is cost effective and rapid. Genotyping technologies supported by the Core include analysis of dinucleotide repeat (microsatellite) markers and single nucleotide polymorphisms (SNPs). These are performed as custom assays using the ABI 3730 capillary DMA analyzer for microsatellite markers, or on the ABI 7900 HT 384-well Real-time PCR machine for Taqman-based SNP genotyping. For whole genome mapping and genetic marker disease association studies, the Core supports microarray analysis of SNPs using both the Affymetrix 10k and 100k SNP genotyping arrays (which query 10,000 or 100,000 polymorphic human genetic markers respectively). In such studies, genomic DNA from affected and unaffected individuals is labeled and hybridized to the genotyping arrays. Using statistical analysis of the genotypes at each of the thousands of markers examined among the control and patient DNA samples, a probability of linkage between the disease trait and each genetic marker (SNP) can be assigned. The Core has supported MRDDRC investigators in mapping genes involved in eye movement disorders and identifying autosomal mutations involved in recessive disorders of brain development. The Affymetrix 10K and 100K SNP genotyping arrays are also being used to map other complex traits such as autism. The expression array component of the facility is an important new addition that has been used extensively by MRDDRC investigators to compare normal and diseased tissue mRNA expression patterns. In these studies, Affymetrix expression arrays are hybridized to probes generated from mRNA isolated from control and diseased tissues. Genes with statistically significant changes in expression (either up- or down-regulated) are identified, and form the basis for new hypothesis driven experiments to understand the molecular basis of the disease. Establishment of this technology was made possible by a Program Project grant to the Core Director. The array component of the Molecular Genetics Core is overseen by Drs. Isaac Kohane and Alan Beggs, and has extensive support from the Children's Hospital bioinformatics group led by Dr. Kohane. Close ties with this program are indicated by 14 collaborative publications between MRDDRC investigators and the bioinformatics group. Historically, the facility has tried to keep up with the latest technologic advances in sequencing, genotyping and expression arrays. Gene expression and SNP genotyping capabilities were recently upgraded with the purchase of the Illumina Bead Array system, which provides users access to customized arrays at lower cost than other platforms. The Core has increased sequencing capacity with the purchase of a second Applied Biosystems 48-capillary sequencer and further increased capacity by implementing an automated workstation for high throughput low volume DNA cycle-sequencing. The Core recently relocated to newly renovated and increased space on the 10th floor of the Enders research building. The staff is highly trained in all the methodologies used within the facility and continue to update their training as new techniques become available. The staff has extensive experience working with MRDDRC investigators in planning experiments and in the analysis of their data. In aggregate, this Core has a strong history and is essential to the research programs of MRDDRC investigators. 6.a.2. Overall Objective The overall objective of the Molecular Genetics Core is to provide high quality, low cost DNA sequencing, microsatellite genotyping and a variety of SNP genotyping and gene expression analysis technologies for MRDDRC investigators. The Core strives to complement these technologies with very strong biostatistics support. It also strives to serve as a reliable resource for investigators pursuing a broader understanding of the genetic basis of developmental disabilities.